Multi-column fractionator

ABSTRACT

A chromatography apparatus for the simultaneous fractionation of two, three and four different mixtures of nucleotides, each on a separate ion-exchange column, using a stepwise elution procedure. Each elution solvent, used in a stepwise elution procedure, is forced by air pressure from a single interchangeable reservoir flask on to and through each of two, three or four chromatography columns at the same moment after having been dispersed equally by a multi-valve control.

United States Patent [1 1 Jones 1 1 MULTl-COLUMN FRACTIONATOR [76]Inventor: Roosevelt J. Jones, 3854 Blodgett,

Houston, Tex. 77004 [22] Filed: Oct. 10, 1973 [21] Appl. No.: 396,356

[52] US. Cl. 210/198 C; 55/386 [51] Int. Cl. B011) 15/08 [58] Field ofSearch 55/386; 210/198 C, 31 C [56] References Cited UNlTED STATESPATENTS 2,935,908 5/1960 Phillips, Jr. 210/198 C X 3,030,798 4/1962Lichtenfels 55/386 X 3,150,517 9/1964 Kuffea et a1. 55/386 X 3,156,54811/1964 Perry 55/386 3,164,980 l/196 5 55/386 X 3,169,389 2/1965 Greenet a1. .1. .z 55/386 X Dec. 16, 1975 3,373,872 3/1968 Habina 210/198 C3,504,799 4/1970 Ogle 210/198 C 3,518,874 7/1970 Habina... 210/198 C X3,653,840 4/1972 Silas 55/386 X Primary Examiner.lohn Adee [57] ABSTRACTA chromatography apparatus for the simultaneous fractionation of two,three and four different mixtures of nucleotides, each on a separateion-exchange column, using a stepwise elution procedure. Each elutionsolvent, used in a stepwise elution procedure, is forced by air pressurefrom a single interchangeable reservoir flask on to and through each oftwo, three or four chromatography columns at the same moment afterhaving been dispersed equally by a multi-valve control. 1

3 Claims, 3 Drawing Figures US. Patent Dec. 16,1975 Sheet10f3 3,926,809

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U.S. Patent Dec. 16, 1975 Sheet 3 of3 3,926,809

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MULTI-COLUMN FRACTIONATOR BACKGROUND OF THE INVENTION This inventionrelates in principle to known liquid 5 ion-exchange chromatographyapparatus (Cohn, 1950. .lour. of Amer. Chem. Soc. 72: 1471-1478) butdiffers in that four columns, rather than one, are employed to permitthe fractionation of four separate and different mixtures of nucleotidesduring a single operation. 19

This apparatus requires a manual placing of each sample or mixture ofnucleotides on to each of four ion-exchange resin columns prior toforcing the elution solvent of a given pH through the resin columns. The

elution solvent of a given pH, when forced through the resin column,will selectively remove certain of the nucleotides of each nucleotidemixture. The apparatus was designed to permit a partial separation ofthe numerous (more than 20) nucleotides which may be present in two,three or four nucleotide mixtures in a rapid fractionator was designedprimarily for the fractionation of Phosphorus-labeled nucleotidemixtures of biological origin where small volumes of several nucleotidemixtures are to be analyzed. To study the synthesis of one or severaldifferent nucleotides, in vivo, as

many as 12 or more biological samples labeled with 30 phosphorus must bequantitatively and qualitatively analyzed within a period of 4 to 6weeks, since the half-life of phosphorus is 14.5 days. An advantage ofthis apparatus is that it permits the simultaneous fractionation of two,three or four different nucleotide 35 mixtures in less time than isrequired to fractionate two different nucleotide mixtures when twoindependent single column chromatography apparatus are employedsuccessively.

SUMMARY This invention is based on the novel concept that the elution ofnucleotide mixtures from different chromatography columns in parallelwould permit the quantitative elution of the same nucleotide when agiven elution solvent and flow rate is employed. This invention wasdevised to permit the elution solvent of a given pH to be forced by airpressure from the reservoir flask through a multi-valve control on toand through each of two, three or four chromatography columns and intocollecting tubes. The interchangeable reservoir flasks providesimplicity and save time during shifting from one elution solvent of onepH to another. A prescribed stepwise elution procedure requires elutingwith several elution solvents of different pH during a single operationor complete fractionation. The multi-valve control makes it possible todisperse equally each elution solvent of a given pH on to and througheach of two, three or four chromatography columns at the same moment.The multi-valve control pennits the regulation of flow rate for eachcolumn. The eluent which flows from each chromatography column iscollected and measured in burettes before transferring into collectingtubes. The collecting of eluent is done manually. The number of elutionsolvents of different pH and the specific pH of each elution solvent canbe prescribed at the discretion of the operator. The size of the resinbed, type of ion-exchange resin used, and

eluent flow rate are not limited to those described herein.

DESCRIPTION OF THE INVENTION FIG. 1 is a schematic four-column liquidchromatography system in accordance with the present invention in oneembodiment.

FIG. 2 shows the component parts of the multicolumn support stand andthe compressed air source viewed from the front.

FIG. 3 is a schematic of the individual components and connections ofthe chromatography apparatus with one of its four chromatography columnsillustrated.

MODE OF OPERATION OF THE INVENTION By way of example and with referenceto the accompanying drawings the embodiments of the present inventionwill now be described. As shown in FIG. 2 the multi-column support standis assembled by fastening, with nut and bolt, the support rod 12 to thesupport stand base 11. The multi-valve support 15 and the column clampsupport 13 are clamped on to the support rod 12. The multi-valve support15 hold the multivalve control 3 in place. The column clamp support 13,with four spring clamps 14, holds the chromatography glass columns 6upright and in place. The ion-exchange resin (Dowex 1X8 formate) isprepared as described by Hurlbert, et al. 1957. .lour. of Biol. Chem.209: 23-39. Each chromatography glass column (28 X 0.8 mm) 6 ispartially filled with the Dowex-l resin (bed size: 15 X 0.5 mm) andrinsed with deionized water prior to and after applying the nucleotidemixture. The nucleotide mixture. The nucleotide mixture can be aphosphorus-labeled nucleotide extract, that contains 100,000 to 800,000counts per minute, or anucleotide mixture, that contains 24 mM ofnucleotides. Stepwise elution of nucleotides, as described by Hurlbert,et al. 1957. .lour. of Biol. Chem. 209: 23-39. involves making severalgraduated shifts in the concentration (from 0.01 M to 4 M formic acid;and 4 M formic acid plus varying concentrations, 0.1 to l M, of ammoniumformate) of the elution solvent during a single operation. The variousnucleotides (mono-, diand triphosphates of the nucleosides of purinesand pyrimidines) which posses different dissociation constants (pKvalues) are eluted individually as a function of the pH of the eluent(Cohn, 1950. Jour. of Amer. Chem. Soc. 72: 1471-1478). The elutionvolume for each concentration of the elution solvent varied from 5 to 20ml between shifts, being collected in 5 ml fractions. To detect thepresence of phosphorus-labeled nucleotides in each eluent fraction a 1ml aliquot is added to 15 ml of a scintillation fluid (POPOP-toluene andTrition X-100, 2:1) and analyzed with a Packard Tri-Carb ScintillationCounter for phosphorus radioactivity. If the eluted nucleotides areunlabeled (containing no phosphorus) their presence in each eluentfraction is determined using a Beckman Model DB Spectre-photometer,which detect ultraviolet-absorbing compounds. Final separation andidentification of individual nucleotides, present in each eluentfraction or in pooled fractions, are accomplished by paperchromatography (Wyatt, 1955. The Nucleic Acids of Chemistry and Biology,Acad. Press, New York. Vol. 1: 243-265) or thin-layer chromatography(Randerath and Randerath, 1964. .Iour. of Chromatography 16: 111-125.

To initiate the elution of nucleotides from the resin columns thereservoir jar 1 containing 400 ml of 0.01

M formic acid is connected to the compressed air tank '18 via the inletglass tube 2B and plastic tubing 2, and to the multi-valve control 3 viathe outlet glass tube 2B and a plastic tubing 2. The dripmeters 5' aredisconnected from the chromatography glass columns 6 before air pressureis permitted to force the elution mixture from the reservoir jar 1,through the multi-valve control 3 and into the dripmeters 5. Adjustmentof the gas regulator 17 and the valve control knobs l6 regulate the flowof the elution solvent. Using the dripmeters 5 the space above the resinbed of each chromatography glass column 6 is filled with the elutionmixture before dripmeters 5 are fitted tightly into each chromatographyglass column 6. Final adjustment with the valve control knobs 16 isnecessary to obtain the desired eluent flow rate of 0.75 ml per minute.Eluent volume is measured in a burette 7, which fits into a buretteplastic cap 8, and collected in test tubes 9, which are held upright intest tube holders l0.

To shift from one elution solvent to the next elution solvent, whose pHconcentration is higher, the following steps are followed. Air pressureis turned off with the gas regulator l7 before disconnecting the plastictubing 2 that joins the gas regulator 17 to the reservoir jar l. Thedripmeter's 5 are disconnected from the chromatography glass columns 6and held elevated above the multi-valve support to permit the elutionsolvent to flow back into the reservoir jar 1 once the valve outlets 19have been opened using the valve control knobs 16. The previously usedreservoir jar 1 is replaced with a reservoir jar 1 containing 400 ml ofelution solvent of the next higher pH concentration. The elution solventabove the resin bed in the chromatography glass columns 6 is removed andreplaced with the elution solvent of the next higher pH concentration.

The elution procedure is resumed after replacing each drip-meter 5 intotheir respective chromatography glass columns 6. The presence of theplastic joint connector 4 which joins the connecting tube 2, leading 5from the valve outlet 19, and the dripmeter 5 permits disconnection ofthe apparatus at this point.

What is claimed is:

1. In an apparatus for analyzing samples of nucleotides using aplurality of chromatograpic columns the improvement comprising incombination:

interchangeable reservoir means. each said reservoir holding an elutionsolvent of a different ph concentration;

a set of four chromatographic columns; a four-way multi-valve controlwhich disperses equally elution solvent of the same pH concentration onto each of said four chromatography columns at the same moment;

said columns containing ion-exchange resin capable of separating amicture of nucleotides; means connecting said valve between saidreservoir and said columns; a burette for measuring eluentvolumecollected from each of said chromatographic columns; a set ofcollecting tubes held upright in test tube holder means; pressure meansconnected to said reservoir means for forcing said elution solventthrough said valve means and into each of said columns. 2 The apparatusof claim 1 wherein said reservoir means consist of wide mouth jar, clampand rubber stopper.

3. The apparatus of claim 2 further including said columns, jar andtubes being constructed of glass.

1. IN AN APPARATUS FOR ANALYZING SAMPLES OF NUCLEOTIDES USING APLURALITY OF CHROMATOGRAPIC COLUMNS THE IMPROVEMMENT 4 INTERCHANGEABLERESERVOIR MEANS, EACH SAID RESERVOIR HOLDING AN ELUTION SOLVENT OF ADIFFERENT PH CONCENTRATION; A SET OF FOUR CHROMATOGRAPHIC COLUMNS; AFOUR-WAY MULTIVALVE CONTROL WHICH DISPERSE EQUALLY ELUTION SOLVENT OFTHE SAME PH CONCENTRATION ON TO EACH OF SAID FOUR CHROMATOGRAPHY COLUMNSAT THE SAME MOMENT; SAID COLUMNS CONTAINING ION-EXCHANGE RESIN CAPABLEOF SEPARATING A MICTURE OF NUCLEOTIDES; MEANS CONNECTING SAID VALVEBETWEEN SAID RESERVOIR AND SAID COLUMNS; A BURETTE FOR MEASURING ELUENTVOLUME COLLECTED FROM EACH OF SAID CHROMATOGRAPHIC COLUMNS; A SET OFCOLLECTING TUBES HELD UPRIGHT IN TEST TUBE HOLDER MEANS; PRESSURE MEANSCONNECTED TO SAID RESERVOIR MEANS FOR FORCING SAID ELUTION SOLVENTTHROUGH SAID VALVE MEANS AND INTO EACH OF SAID COLUMNS.
 2. The apparatusof claim 1 wherein said reservoir means consist of wide mouth jar, clampand rubber stopper.
 3. The apparatus of claim 2 further including saidcolumns, jar and tubes being constructed of glass.